Survey of potential root pathogens in softwood cuttings collected from Georgia blueberry nurseries

Survey sampling of 18 blueberry nurseries propagating softwood cuttings was conducted in 2007 and 2008 to determine which soilborne plant pathogens were most prevalent in commercial blueberry propagation systems in southern Georgia. Samples were collected four times: June (early), September (mid), and October (late) in the 2007 growing season, with additional sampling of overwintered cuttings in April 2008. This survey revealed that Calonectria ilicicola (Cylindrocladium parasiticum), causing Cylindrocladium root rot, is a primary pathogen of blueberry cuttings in southern Georgia. The organism was isolated consistently during all four survey dates with a cutting-level incidence of 3.6, 10.2, 36.4, and 14.3% in the first through fourth samplings, respectively. Rhizoctonia spp. were recovered less frequently and were only present during the first (10.9% cutting-level incidence), third (4.6%), and fourth survey dates (3.6%), while the Oomycetes Pythium and Phytophthora were detected only on the second survey date. Fusarium spp. were isolated commonly (29.1% incidence in the first, 12.2% in the second, 18.2% in the third, and 7.1% in the fourth sampling), but pathogenicity remains uncertain. Across all survey dates, Calonectria, Rhizoctonia, Oomycetes, and Fusarium were recovered at least once from 41.2, 17.6, 0.1, and 82.4% of nurseries surveyed, respectively. When nursery-level pathogen presence-absence data collected from this survey was analyzed in relation to a survey of production practices used by these same propagators, the practice of reusing growth media was found significantly associated with Calonectria presence.

Effect of Container Size, Inoculum Density, and Test Duration on Detecting Resistance to Cylindrocladium Black Rot of Peanut

Cylindrocladium black rot (CBR), caused by the fungal pathogen Cylindrocladium parasiticum Crous, Wingfield, and Alfenas, is an important peanut (Arachis hypogaea L.) disease in the Virginia-Carolina and Southeastern U.S. production regions and was first confirmed in Texas in 2004. We refined a screening technique and compared disease assessment methods for CBR using peanut germplasm with known resistance levels. Resistant genotype ‘NC 3033’ and susceptible ‘NC 7’ were compared in a growth chamber using 66 and 164 cm3 container sizes, 15 and 25 microsclerotia/g soil inoculum densities, and 4, 5, and 6 week durations. Root rot ratings (0 to 5 index), percent taproot necrosis and percent secondary root necrosis were estimated. Taproot necrosis was the most reliable method for differentiating between the resistant and susceptible genotype, followed by root rot ratings. The use of secondary root necrosis ratings was less consistent for detecting differences than taproot ratings. Large containers (164 cm3) inoculated at the 25 microsclerotia/g soil density was the most reliable combination for attaining significant genotype differences for taproot necrosis and root rot ratings. The 5 week duration provided the most consistent results for taproot necrosis, while 6 weeks was the most reliable duration for root rot ratings. Percent taproot necrosis is a valid, more objective alternative to root rot index ratings.

First Report of Cylindrocladium Black Rot of Peanut Caused by Cylindrocladium parasiticum (Teleomorph Calonectria ilicicola) in Jiangxi Province, China

In July 2010, a serious disease of peanut (Arachis hypogaea) resembling Cylindrocladium black rot (CBR) was found in Longnan County, Jiangxi Province, China. Symptoms included chlorotic, yellowish and blighted leaves, and wilting of the plants. Taproots and hypocotyls were blackened and rotted. Clusters of reddish orange spherical fruiting bodies appeared in the lesions present on basal stems, pegs, pods, and roots of peanut. Disease incidence reached as much as 50% in some patches of the field. Plants with symptoms were sampled from fields. Microscopic examination revealed that the reddish orange, spherical fruiting bodies were the perithecia and measured 461.6 (337.5 to 609.4) × 395.5 (309.4 to 496.9) μm. With gentle pressure, asci and ascospores were exuded from perithecia. The asci were hyaline, thin walled, and long stalked. Ascospores were hyaline, falcate with one septum, and measured 43.5 (27.3 to 54.5) × 5.6 (4.1 to 6.8) μm with a length/width (L/W) ratio of 7.8 ± 1.3. A fungus with white-to-pale buff border mycelia and yellowish brown pigment was consistently isolated from the edge of basal stem lesions on potato dextrose agar at 25°C. Mycelia grew at temperatures ranging from 8 to 32°C and the optimum was 25 to 26°C. To determine the species, single-conidial isolates of the fungus were cultured on carnation leaf agar for 7 days at 25°C and 12 h of light/dark conditions. Conidia were hyaline, cylindrical with one to three septa (mostly three septa), and measured 49.3 (27.3 to 70.9) × 5.9 (4.1 to 6.8) μm with L/W ratio of 8.4 ± 1.6. Vesicles were globose and measured 5.5 to 10.9 μm in diameter. The fungus was identified as Cylindrocladium parasiticum (teleomorph Calonectria ilicicola) (1,2). A PCR assay was conducted on one representative isolate (JXLN32) by analyzing multilocus sequences of the TUB2 (coding β-tubulin protein), ACT (coding actin), and CaM gene (coding calmodulin protein) and were amplified and sequenced using the primers reported by Crous et al. (3). Sequences of the studied DNA regions were submitted to GenBank (Accession Nos. TUB2: JF429649; ACT: JQ070809; and CaM: JQ070808). BLAST searches with the existing sequences in GenBank showed that there was 99 to 100% identity with the existing sequences of C. ilicicola (GenBank Accession Nos. TUB2: AY725643; ACT: GQ280446; and CaM: GQ267402). To complete Koch's postulates, inoculum was prepared by mixing the microsclerotia (MS) suspension of the isolate (JXLN32) with soil at a proportion of 10 MS per g of soil. Ten replicate plastic pots containing five peanut seeds (cv. Yueyou 7) each were planted and placed in a glasshouse at 25 ± 2°C. The same number of peanut seeds was used as an uninoculated control. Typical basal stem and roots rot symptoms of CBR were observed in 2 months and C. parasiticum was reisolated from these inoculated diseased plants. No symptoms were detected on the control plants. To our knowledge, this is the first finding of Cylindrocladium black rot in Jiangxi Province, which is the main peanut-producing area in China. The disease has been previously reported in Guangdong Province in southern China but is not known elsewhere (4). Because of its ability to spread through seed and soil and its destructive potential, this pathogen may pose a serious threat to peanut production in China. References: (1) D. K. Bell and E. K. Sobers. Phytopathology 56:1361, 1966. (2) P. W. Crous et al. Mycol. Res. 97:889, 1993. (3) P. W. Crous et al. Stud. Mycol. 50:415, 2004. (4) R. Pan et al. Plant Pathol. 58:1176, 2009.

First Report of Peanut Cylindrocladium Black Rot Caused by Cylindrocladium parasiticum in Fujian Province, Eastern China

During late July and early August of 2010, a serious disease of peanut (Arachis hypogaea L.) resembling Cylindrocladium black rot (CBR) was found in Longyan City, Fujian Province of Eastern China. Aboveground symptoms were chlorosis and yellowing of leaves, a black rot of the basal stem and pegs, and wilting of the vines. Underground plant parts (including pods, pegs, taproots, and hypocotyls) were blackened and rotted. Orange-to-reddish spherical fruiting bodies appeared on the lesions of the basal stems and pegs of peanut. Disease incidence was approximately 20%. A fungus was consistently isolated from the edge of lesions on potato dextrose agar (PDA) amended with streptomycin and incubated at 25°C. The fungus produced white-to-pale buff mycelia with a yellowish brown pigment. Optimum growth of the fungus on PDA was at 25 to 30°C. Conidiophores were borne laterally on a stipe that terminated in a hyaline, globose vesicle measuring 5.5 × 10.9 μm in diameter. Conidia were hyaline, cylindrical, rounded at both ends, slightly wider at the base than at the apex, with one to three septa (mostly one septa), and measured 27.3 to 70.9 × 4.1 to 8.2 μm. Orange-to-reddish perithecia were readily formed in old cultures. The perithecia were subglobose to oval or obovate and measured 215.6 to 609.4 × 309.4 to 496.9 μm. The asci were hyaline, clavate, thin walled, long stalked, with each containing eight ascospores. Ascospores were hyaline, falcate, had one septum, and measured 27.3 to 54.5 × 4.1 to 6.8 μm. The fungus was identified as Cylindrocladium parasiticum Crous, M.J. Wingfield, & Alfenas (teleomorph Calonectria ilicicola) (1,2). The beta-tubulin gene fragment was amplified using the T1/Bt2b primers (3) and sequenced. The sequences of three isolates (GenBank Accession Nos. JF343965, JF429656, and JF429657), when compared with existing sequences in GenBank, had 95 to 99% sequence identity with Calonectria ilicicola (GenBank Accession Nos. AY725643 and AY725639). Pathogenicity tests were conducted by first culturing the fungus on wheat kernels for 2 weeks. Inoculated kernels were then used as inoculum and mixed with sterilized soil in a proportion of 1:20 by weight in plastic pots (10 × 9 cm). Noninoculated wheat kernels were mixed with sterilized soil in the same proportion and served as the control. Two-week-old peanut seedlings (cv. Yueyou No. 7) were transplanted into inoculated or noninoculated pots. There were five plants per pot and each treatment was replicated four times. The plants were incubated in a greenhouse at 25 ± 2°C. All of the treated plants exhibited typical basal stem and root rot symptoms of CBR 2 weeks after inoculation, while all of the control plants remained healthy. C. parasiticum was reisolated from the diseased plants. To our knowledge, this is the first report of CBR on peanut in Fujian Province in Eastern China. The disease has been previously reported in Guangdong Province in Southern China but is not known elsewhere (4). This pathogen may pose a serious threat to peanut production in China, where peanut is an important crop. References: (1) D. K. Bell and E. K. Sobers. Phytopathology 56:1361, 1966. (2) P. W. Crous et al. Mycol. Res. 97:889, 1993. (3) P. W. Crous et al. Can. J. Bot. 77:1813, 1999. (4) R. Pan et al. Plant Pathol. 58:1176, 2009.

New Sources of Cylindrocladium Black Rot Resistance among Runner-Type Peanut Cultivars

Cylindrocladium Black Rot (CBR) caused by Cylindrocladium parasiticum Crous, Wingfield, & Alfenas syn. C. crotalariae (Loos) Bell & Sobers is a major disease problem in southeast U.S. peanut (Arachis hypogaea L.) production. Field trials were conducted for two-years (2008-09) at a test site (Gibbs Farm) that has a long history of continuous peanut production near the Univ. of Georgia, Coastal Plain Expt. Station, Tifton, GA to evaluate for CBR resistance among new runner-type peanut cultivars. All plots were artificially inoculated with microsclerotia of C. parasiticum after planting each year. Significant differences (P≤0.05) were found among the cultivars and advanced breeding lines for both CBR resistance and tomato spotted wilt virus (TSWV) resistance which was also present each year, but the predominant disease was CBR. Georgia Greener, Georgia-06G, Georgia-07W, Georgia-02C, and Carver were consistently found to be the most CBR resistant; whereas, C-99R and Tifguard were the most susceptible each year. In separate CBR tests conducted in 2009 and 2010 at a different location (Blackshank Farm), Georgia Greener also had the least difference, and Tifguard had the greatest difference, between C. parasiticum inoculated versus non-inoculated plots for pod yield. These combined test results demonstrate that useful levels of CBR resistance are currently available in promising new runner-type peanut cultivars.

First Report of Neocosmospora striata Causing Peanut Pod Rot in China

In 2008, an outbreak of pod rot of peanut (Arachis hypogaea L.) occurred on most of the peanut cultivars in the Old Yellow River drainage area, the largest peanut-growing region in China. Disease incidence reached as high as 90% in some fields, causing severe yield losses. The black rot of pods and blackened, nonrotting taproots is similar to symptoms of peanut black rot caused by Cylindrocladium parasiticum, but the reddish orange perithecia of C. parasiticum were not found on the taproots close to the surface of the soil. The foliage of affected plants was generally asymptomatic, but some plants turned greener. This pod rot disease was further investigated in 2008 and 2010. Twenty-three Fusarium-like isolates were obtained from symptomatic, surface-disinfested pods with a frequency of 82%. These isolates were fast growing, with flat, thin, and grayish white colonies when cultured on potato dextrose agar (PDA) at 28°C for 3 to 4 days. The hyaline, elongated to cylindrical conidia, aggregated in slimy heads on conidiogenous cells developed from undifferentiated hyphae when observed with the light microscope. The size of conidia (single celled or one septum) varied from 3 to 9 μm long and 1.5 to 3.5 μm wide on the basis of the measurement of 50 spores. Some conidia appeared slightly curved. Ascomata formed within 10 to 14 days, with a punctate appearance on the colony. The cerebriform ascomata were dark brown, pyriform, ostiolate, glabrous, 120 to 170 × 90 to 130 μm, and with necks 30 to 50 μm long. Asci measured 60 to 90 × 6 to 10 μm, were cylindrical to cylindric-clavate, thin walled, and had an apical ring. Ascospore arrangement was obliquely uniseriate or partially biseriate, very pale yellow to hyaline, ellipsoidal, and measured 8 to 12 × 4.5 to 6 μm. Some spores had a median transverse straight or curved septum and were slightly constricted at the septum, with 6 to 10 thin, transverse, hyaline flanges. Morphological characteristics of the isolates with ascomata dark brown and ascospores with 6 to 10 transverse hyaline flanges matched the description for Neocosmospora striata (1). The internal transcribed spacer (ITS) region of rDNA was amplified from extracted template DNA with primer pairs ITS4/ITS5 and sequenced. A 591-bp amplicon (GenBank Accession No. HM461900) had 99% sequence identity with Fusarium solani (HQ607968 and HQ608009) and N. vasinfecta (GU213063), which indicated that these fungi belong to the genus Neocosmospora or Fusarium, although there is no direct sequence evidence that they are N. striata. N. striata has only been previously reported in Japan (2). This species is unique because of the dark brown ascomata and there is no comparable species (1). Koch's postulates were completed by surface-disinfesting 80 peanut pods of cv. Jihua 9813 and soaking them in conidial suspensions (105 conidia/ml) for 2 min. Another 80 other pods soaked in sterile water served as controls. All peanuts were incubated in moist petri dishes under darkness at 28°C. Symptoms similar to those originally observed in the field formed within 10 days on all inoculated peanut pods and not the controls. N. striata was reisolated from all affected peanut pods. To our knowledge, this is first report of N. striata causing peanut pod rot in China and the first description of the anamorph of the fungus. References: (1) P. F. Cannon et al. Trans. Br. Mycol. Soc. 82:673, 1984. (2) S. Udagawa et al. Trans. Mycol. Soc. Jpn. 16:340, 1975.